DESCRIPTION (Applicant's Abstract): This program will evaluate the properties of a novel glutathione peroxidase enzyme that is considerably enriched in lungs, especially granular pneumocytes and bronchiolar epithelium. This recently described protein (called l-cysPeroxiredoxin or 1-cysPrx) does not contain selenium and thus represents the first non-selenium gluthathione peroxidase of relatively high specific activity. An important characteristic in the spectrum of activity for this enzyme is its ability to reduce phospholipid hydroperoxides, unlike classical glutathione peroxidase. We postulate that 1-cysPrx can reduce oxidized phospholipids in membranes and hypothesize that it is an important pulmonary antioxidant enzyme. We have previously described methods for isolation of l-cysPrx protein from rat and bovine lungs and have published the human, rat, mouse and bovine cDNA sequences. We have found that mutation of Cys 47 (the only conserved Cys residue) abolishes peroxidase activity with H2O2 and phospholipid hydroperoxide substrates compatible with this as the active site. An important preliminary funding is that site-directed mutagenesis of Ser 32 has no effect on reduction of H2O2 but abolishes activity with phospholipid hydroperoxide substrates indicating importance of secondary recognition/binding sites for phospholipid hydroperoxides. Based on this preliminary data and the crystal structure, we have developed a model of enzyme activity which requires a dimeric (or tetrameric) protein with a Ser 32 recognition site, a hydrophobic pocket for phospholipid substrate binding, and a Cys 47 active site. Specific Aim 1 will evaluate physical properties of 1-cysPrx with special emphasis on the oligomeric state required for enzymatic activity. Specific Aim 2 will evaluate our proposed model for substrate binding by testing hydroperoxide substrates and phospholipid liposomes with native and mutant proteins using tryptophan fluorescence and circular dichroism measurements. Specific Aim 3 will evaluate specific activity of native and mutant enzyme preparations in order to test our predictions concerning the physical basis for enzymatic activity. Specific Aim 4 will evaluate the role of 1-cysPrx in antioxidant defense using both in vitro and in vivo approaches. Cell lines that overexpress 1-cysPrx will be generated and evaluated for resistance to oxidative stress (H2O2, paraquat). To study tolerance in vivo, rats and mice will be exposed to hyperoxia; enzyme induction (mRNA, protein, activity) will be measured in control and selenium deficient rats exposed to 80 percent or 100 percent 02 and in mice with knock out of classical glutathione peroxidase. A mouse model with knock out of l-cysPrx currently under development will also be tested for oxygen tolerance. These studies will give insights into the function and antioxidant activity of this potentially important, newly described enzyme.